Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma Cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Charge generation and recombination in solid-state dye-sensitized solar cells

Diese Doktorarbeit befasst sich mit Ladungsgeneration und – rekombination in Feststoff-Farbstoffsolarzellen, die spiro-OMeTAD als Lochleiter verwenden. Die vorliegende Arbeit ist in drei Fallstudien unterteilt: i.) Kern-erweiterte Rylen-Farbstoffe, ii.) ein Perylenmonoimid-Farbstoff und iii.) Donor-π verbrückte (Cyclopentadithiophen)-Akzeptor-Farbstoffe. Trotz ihres hohen molaren Extinktionskoeffizienten und der hohen Absorbanz der sensibilisierten Filme, zeigen einige dieser Farbstoffmoleküle nur geringe photovoltaischen Effizienzen. Um den Ursprung des geringen Wirkungsgrades herauszufinden, wurde breitbandige, ultraschnelle transiente Absorptionsspektroskopie an Solarzellen durchgeführt.rnInsbesondere die Auswirkungen verschiedender Ankergruppen, Dipolmomente, Photolumineszenzlebenszeiten, Lithium-Kationensensitivität und Ladungsträgerdynamik, die alle einen großen Einfluss auf den Wirkungsgrad der Solarzelle besitzen, wurden untersucht. In der ersten Fallstudie zeigte ein kurzer Rylen-Farbstoff aufgrund deutlich verlängerter Lebenszeiten die beste Effizienz im Vergleich zu größeren Kern-erweiterten Rylen-Farbstoffen. Die Lebenszeit wurde weiter reduziert, wenn Maleinsäure als Ankergruppe unter einer Ringöffnungsreaktion an die mesoporöse Oberfläche des Metalloxid-Halbleiters adsorbierte. Dies konnte mit Hilfe von Berechnungen mittels der Dichtefunktionaltheorie (DFT, B3LYP) auf die Differenz des Dipolmoments zwischen Grundzustand und angeregtem Zustand zurückgeführt werden. Die Berechnungen bekräftigen die unvorteilhafte Injektion von Ladungen durch die Änderung der Richtung des Dipolmoments, wenn eine Ringöffnung der Anhydridgruppe stattfindet. In der zweiten Studie zeigte das Perylenmonoimid-Derivat ID889 einen Wirkungsgrad von 4.5% in Feststoff-Farbstoffsolarzellen, wobei ID889 sogar ohne Zuhilfenahme eines Additivs in der Lage ist langlebige Farbstoffkationen zu bilden. Die Verwendung von Lithium-Kationen stabilisiert jedoch sowohl den Prozess der Ladungsgeneration als auch den der Ladungsregeneration. Des Weiteren wurde in ID889-sensitivierten Bauteilen kein reduktives Löschen beobachtet. Dabei wurde die Dynamik der Exzitonen mittels einer soft-modelling Methode Kurvenanalyse aus den Daten der transienten Absorptionsspektroskopie gewonnen. Zuletzt wurden Strukturen mit Cyclopentadithiophen(CPDT)-Baustein untersucht, die eine typische D-π-A Molekülstruktur bilden. FPH224 und 233 zeigten dabei eine bessere Effizienz als FPH231 und 303 aufgrund einer großen Injektionseffizienz (IE) und längerer Lebenszeit der angeregten Zustände. Dies kann auf reduktives Löschen in FPH231 und 303 zurückgeführt werden, wohingegen FPH224 und 233 einen moderaten Zerfall des Spirokationensignals zeigten.

Simulated switching of the resting state functional connectivity in mouse brain using a real mesoscale connectome

Capire come modellare l'attività del cervello a riposo, resting state, è il primo passo necessario per avvicinarsi a una reale comprensione della dinamica cerebrale. Sperimentalmente si osserva che, quando il cervello non è soggetto a stimoli esterni, particolari reti di regioni cerebrali presentano un'attività neuronale superiore alla media. Nonostante gli sforzi dei ricercatori, non è ancora chiara la relazione che sussiste tra le connessioni strutturali e le connessioni funzionali del sistema cerebrale a riposo, organizzate nella matrice di connettività funzionale. Recenti studi sperimentali mostrano la natura non stazionaria della connettività funzionale in disaccordo con i modelli in letteratura. Il modello implementato nella presente tesi per simulare l'evoluzione temporale del network permette di riprodurre il comportamento dinamico della connettività funzionale. Per la prima volta in questa tesi, secondo i lavori a noi noti, un modello di resting state è implementato nel cervello di un topo. Poco è noto, infatti, riguardo all'architettura funzionale su larga scala del cervello dei topi, nonostante il largo utilizzo di tale sistema nella modellizzazione dei disturbi neurologici. Le connessioni strutturali utilizzate per definire la topologia della rete neurale sono quelle ottenute dall'Allen Institute for Brain Science. Tale strumento fornisce una straordinaria opportunità per riprodurre simulazioni realistiche, poiché, come affermato nell'articolo che presenta tale lavoro, questo connettoma è il più esauriente disponibile, ad oggi, in ogni specie vertebrata. I parametri liberi del modello sono stati scelti in modo da inizializzare il sistema nel range dinamico ottimale per riprodurre il comportamento dinamico della connettività funzionale. Diverse considerazioni e misure sono state effettuate sul segnale BOLD simulato per meglio comprenderne la natura. L'accordo soddisfacente fra i centri funzionali calcolati nel network cerebrale simulato e quelli ottenuti tramite l'indagine sperimentale di Mechling et al., 2014 comprovano la bontà del modello e dei metodi utilizzati per analizzare il segnale simulato.

Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches

Please cite this paper as: PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches. Experimental Dermatology 2010. Abstract: Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50-60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high-resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin-fixed and paraffin-embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low-level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short-time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long-time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.

Genome-wide association study of intracranial aneurysm identifies three new risk loci

Saccular intracranial aneurysms are balloon-like dilations of the intracranial arterial wall; their hemorrhage commonly results in severe neurologic impairment and death. We report a second genome-wide association study with discovery and replication cohorts from Europe and Japan comprising 5,891 cases and 14,181 controls with approximately 832,000 genotyped and imputed SNPs across discovery cohorts. We identified three new loci showing strong evidence for association with intracranial aneurysms in the combined dataset, including intervals near RBBP8 on 18q11.2 (odds ratio (OR) = 1.22, P = 1.1 x 10(-12)), STARD13-KL on 13q13.1 (OR = 1.20, P = 2.5 x 10(-9)) and a gene-rich region on 10q24.32 (OR = 1.29, P = 1.2 x 10(-9)). We also confirmed prior associations near SOX17 (8q11.23-q12.1; OR = 1.28, P = 1.3 x 10(-12)) and CDKN2A-CDKN2B (9p21.3; OR = 1.31, P = 1.5 x 10(-22)). It is noteworthy that several putative risk genes play a role in cell-cycle progression, potentially affecting the proliferation and senescence of progenitor-cell populations that are responsible for vascular formation and repair.

Local strain distribution in real three-dimensional alveolar geometries

Mechanical ventilation is not only a life saving treatment but can also cause negative side effects. One of the main complications is inflammation caused by overstretching of the alveolar tissue. Previously, studies investigated either global strains or looked into which states lead to inflammatory reactions in cell cultures. However, the connection between the global deformation, of a tissue strip or the whole organ, and the strains reaching the single cells lining the alveolar walls is unknown and respective studies are still missing. The main reason for this is most likely the complex, sponge-like alveolar geometry, whose three-dimensional details have been unknown until recently. Utilizing synchrotron-based X-ray tomographic microscopy, we were able to generate real and detailed three-dimensional alveolar geometries on which we have performed finite-element simulations. This allowed us to determine, for the first time, a three-dimensional strain state within the alveolar wall. Briefly, precision-cut lung slices, prepared from isolated rat lungs, were scanned and segmented to provide a three-dimensional geometry. This was then discretized using newly developed tetrahedral elements. The main conclusions of this study are that the local strain in the alveolar wall can reach a multiple of the value of the global strain, for our simulations up to four times as high and that thin structures obviously cause hotspots that are especially at risk of overstretching.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

3CAM - A NOVEL TERNARY CONTENT ADDRESSABLE MEMORY USING THREE-VALUED LOGIC

Content Addressable Memory (CAM) is a special type of Complementary Metal-Oxide-Semiconductor (CMOS) storage element that allows for a parallel search operation on a memory stack in addition to the read and write operations yielded by a conventional SRAM storage array. In practice, it is often desirable to be able to store a “don’t care” state for faster searching operation. However, commercially available CAM chips are forced to accomplish this functionality by having to include two binary memory storage elements per CAM cell,which is a waste of precious area and power resources. This research presents a novel CAM circuit that achieves the “don’t care” functionality with a single ternary memory storage element. Using the recent development of multiple-voltage-threshold (MVT) CMOS transistors, the functionality of the proposed circuit is validated and characteristics for performance, power consumption, noise immunity, and silicon area are presented. This workpresents the following contributions to the field of CAM and ternary-valued logic:• We present a novel Simple Ternary Inverter (STI) transistor geometry scheme for achieving ternary-valued functionality in existing SOI-CMOS 0.18µm processes.• We present a novel Ternary Content Addressable Memory based on Three-Valued Logic (3CAM) as a single-storage-element CAM cell with “don’t care” functionality.• We explore the application of macro partitioning schemes to our proposed 3CAM array to observe the benefits and tradeoffs of architecture design in the context of power, delay, and area.